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1、時相點問題，比較mRNA合理點?，F(xiàn)象：IL-1Ra 3h 開始表達。分析：影響因素有哪些？ABDECFFig. 1Poly(I-C) induced IL-1Ra production by stimulating TLR3 on normal synovial fibroblasts (FLS) through IRF3 IRF3 regulates the transcription of IL-1Ra in response to TLR3 activation in normal synovial fibroblasts (FLS) ABFigure 1. Poly(I-C) indu

2、ced IL-1Ra production on normal synovial fibroblasts (FLS)dsRNACDABCDS1. Poly(I-C) induced IL-1Ra production on normal FLSPoly(I:C) :-+IL-1RaGAPDH17KD37KDABDECFFigure 2. Poly(I-C) induced IL-1Ra production through TLR3 補充TLR3knockout或Trif knockout的結(jié)果MDA5和RIG1 knockdown5ppp RNA RIG1Many studies invol

3、ve the use of poly I:C as a surrogate for natural dsRNA in the stimulation of for TLR3. Although certainly helpful, such results need to be interpreted with some caution as different commercially available poly I:C compounds exert somewhat differential effects, e.g., interleukin (IL)-12p70 induction

4、 in DCs, probably dependent on differences in molecular weight and the fact that it is not a physiologically or even naturally occurring substance. Also, stimulation of additional receptors including cytosolic Mda5 and RIG-I has been observed.(Not all polyriboinosinic-polyribocytidylic acids (Poly I

5、:C) are equivalent for inducing maturation of dendritic cells: implication for alpha-type-1 polarized DCs,2009,Journal/J Immunother)Of note, mammalian mRNA, i.e., self-RNA has also been postulated to be a TLR-3 ligand,(mRNA is an endogenous ligand for Toll-like receptor 3,2004,Journal/J Biol Chem) w

6、hich might signal the presence of necrotic debris from neighboring cells. Possibly, structured elements, especially in the UTRs of mRNA might form secondary structures that could induce TLR-3 signaling.From (RNA mediated Toll-like receptor stimulation in health and disease,2012,Journal/RNA biology)

7、Poly(I:C) :-+ -+HCQ :-+Poly(I:C) :-+ -+HCQ :-+Poly(I:C) :-+ -+HCQ :-+Poly(I:C) :-+ -+siRNA TLR3 :-+Poly(I:C) :-+ -+siRNA TLR3 :-+Poly(I:C) :-+ -+siRNA TLR3 :-+ABS2. Production of IL-1Ra was independent of interferon-(IFN-) Poly(I:C) :-+-Anti-IFN- mAb :-+-+IFN- :-+IFN-IL-1RaPalmer, G, et al. Ann Rheu

8、m Dis. 2004Darragh, J., et al. Biochem J. 2010ABCDFigure 4. Production of IL-1Ra was independent of de novo protein synthesisPoly(I:C) :-+-+Cycloheximid:-+Poly(I:C) :-+-+Cycloheximid:-+Poly(I:C) :-+-+Cycloheximid:-+Poly(I:C) :-+-+Cycloheximid:-+Goh, F. G. and K. S. Rheumatology (Oxford). 2012ABDECFF

9、igure 5. Production of IL-1Ra was dependent of IRF3 Inhibition of IL-1Ra(WB)EMSA ( Electrophoretic Mobility Shift Assay ): IRF3 binding with IL-1Ra promoterIRF3 knockoutPoly(I:C) :-+ -+siRNA IRF3 :-+Poly(I:C) :-+ -+siRNA IRF3 :-+Poly(I:C) :-+ -+siRNA IRF3 :-+Sarkar, S. N., et al. Nat Struct Mol Biol

10、. 2004ABDECFFigure 6. Production of IL-1Ra was dependent of PI3KIFN-Poly(I:C) :-+-+LY294002 :-+GAPDHPoly(I:C) :-+LY294002:-+Phospho-AktPoly(I:C) :-+-+LY294002 :-+Poly(I:C) :-+-+LY294002 :-+Poly(I:C) :-+-+LY294002 :-+ABDECFFigure 7. Production of IL-1Ra was dependent of GSK3IFN-Poly(I:C) :-+-+SB41528

11、6 :-+GAPDHPoly(I:C) :-+SB415286:-+Phospho-GSK3Poly(I:C) :-+-+SB415286 :-+Poly(I:C) :-+-+SB415286 :-+Poly(I:C) :-+-+SB415286 :-+Darragh, J., et al. Biochem J. 2010ABDECFFigure 8. Production of IL-1Ra was dependent of NF-BInhibition of IL-1Ra(WB)EMSA ( Electrophoretic Mobility Shift Assay ) : NF-B bin

12、ding with IL-1Ra promoterPoly(I:C) :-+-+BMS-345541:-+GAPDHPoly(I:C) :-+BMS-345541:-+Phospho-IBPoly(I:C) :-+-+BMS-345541:-+Poly(I:C) :-+-+BMS-345541:-+Poly(I:C) :-+-+BMS-345541:-+ABDECFFigure 9. Production of IL-1Ra was negative regulated by ERKPoly(I:C) :-+-+PD184352 :-+GAPDHPoly(I:C) :-+PD184352:-+

13、p-ERK 1/2Poly(I:C) :-+-+PD184352 :-+Poly(I:C) :-+-+PD184352 :-+Poly(I:C) :-+-+PD184352 :-+Darragh, J., et al. Biochem J. 2010Darragh, J., et al. Biochem J. 2010ABDECFFigure 10. Production of IL-1Ra was negative regulated by MSK1/2Poly(I:C) :-+ -+siRNA MSK1/2 :-+Poly(I:C) :-+ -+siRNA MSK1/2 :-+p-CREB

14、p-ATF-1LPS:-+Time (min):-1530GAPDHp-CREBp-ATF-1Poly(I:C) :-+Time (min):-1530GAPDHp-MSKp-MSKRAWFLSPoly(I:C) :-+-+SB747651A :-+Poly(I:C) :-+-+SB747651A :-+Poly(I:C) :-+-+SB747651A :-+Darragh, J., et al. Biochem J. 2010ABDECFFigure 11. Production of IL-1Ra was independent of p38 MAPKPoly(I:C) :-+-+SB203580 :-+GAPDHPoly(I:C) :-+SB203580:-+Phosp